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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 June 2010 to 10 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: pending to search
Deviations:
not applicable
Principles of method if other than guideline:
The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 Hhour post exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end point will be used to either confirm a non-irritant result or will be used to override the non irritant result.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): BAB
- Substance type: UVCB
- Physical state: clear colourless liquid
- Purity test date: 01 June 2010
- Lot/batch No.: 86364/1-10
- Expiration date of the lot/batch: 01 June 2015
- Storage condition of test material: room temperature in the dark

Test animals

Species:
other: not applicable

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 10 µl of the test material was applied to the epidermis surface (to ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre)).
After 7 minutes contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period.
Duration of treatment / exposure:
15 minutes
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
97.8
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. Reversibility: other: not applicable. (migrated information)
Other effects / acceptance of results:
The relative mean viability of the test material treated tissues was 97.8% after a 15 minute exposure.
The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Any other information on results incl. tables

Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material:

Material

OD540 of tissues

Mean OD540 of triplicate tissues

± SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Material

0.687

0.687

0.007

100.0

100*

1.0

0.681

99.1

0.694

101.0

Positive Control Material

0.105

0.067

0.035

15.3

9.7

5.1

0.060

8.7

0.035

5.1

Test Material

0.654

0.672

0.030

95.2

97.9

4.4

0.656

95.5

0.707

102.9

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Following the 15-Minute exposure period the test material treated tissues appeared blue which was considered indicative of viable tissue.

Due to the absence of cytotoxicity in the MTT assay it was necessary to determine the concentration ofinflammatory mediator IL-1α in the retained culture medium for confirmatory purposes.

The concentration ofinflammatory mediator IL-1α in the culture medium retained from the test material treated tissues was 27.273 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤ 18%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥ 0.6 and the SD value of the percentage viability was ≤ 18%. The negative control acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: other: see table above (any other information on materials and methods)
Conclusions:
The test material was considered to be Non-Irritant. This result was confirmed by assessment of the inflammatory mediator IL-1α.
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hhour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 97.8% after a 15-Minute exposure period.

Due to the absence of cytotoxicity in the MTT assay it was necessary to determine the concentration of inflammatory mediator IL-1α in the retained culture medium for confirmatory purposes.

The concentration of inflammatory mediator IL-1α in the culture medium retainedfrom the test material treated tissues was 27.273 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: 

The test material was considered to be Non-Irritant. This result was confirmed by assessment of the inflammatory mediator IL-1α.